Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2000-08-14
2003-01-07
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S005000, C424S188100, C424S148100
Reexamination Certificate
active
06503753
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the fields of virology, immunology, disease treatment and prevention. More particularly, it concerns HIV particles with inactivated reverse transcriptase, methods of inactivation, and the use of such particles to prepare components of HIV and to elicit effective immunological responses to HIV. These immune responses are useful in producing diagnostic reagents, assays, and kits for the diagnosis of HIV and related retroviral disease, providing protection from an HIV challenge, and assisting an HIV-infected individual in controlling the replication of the virus. Methods of inactivation are useful for preventing disease through decreasing the risk of infection associated with exposure to HIV infected tissues and materials.
1.2 Description Related Art
1.2.1 Human Immunodeficiency Virus
Human Immunodeficiency Virus-i (HIV-1) infection has been reported throughout the world in both developed and developing countries. HIV-2 infection is found predominately in West Africa, Portugal, and Brazil. It is estimated that as of 1990 there were between 800,000 and 1.3 million individuals in the United States that were infected with HIV. An important obstacle to developing a vaccine against HIV is that the mechanism of immunity to HIV infection is ill-understood. Not all of those infected individuals will develop acquired immunodeficiency syndrome (AIDS). Indeed recent reports have suggested that there may be certain individuals that are resistant to HIV-1 infection.
The HIV viruses are members of the Retroviridae family and, more particularly, are classified within the Lentivirinae subfamily. Like nearly all other viruses, the replication cycles of members of the Retroviridae family, commonly known as the retroviruses, include attachment to specific cell receptors, entry into cells, synthesis of proteins and nucleic acids, assembly of progeny virus particles (virions), and release of progeny viruses from the cells. A unique aspect of retrovirus replication is the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that must integrate into the genome of the host cell prior to the synthesis of viral proteins and nucleic acids.
Retrovirus virions are enveloped and contain two copies of the genome. The conversion of the genomic RNA into DNA is provided by the viral protein reverse transcriptase (RT). This protein is bound to the RNA genome within the virion, and its enzymatic conversion of the genome to DNA is believed to take place after viral entry into the host cell. However, recent evidence suggests that the conversion process may initiate in the virion particles themselves, known as endogenous reverse transcription (ERT), and that ERT may be important in increasing the infectivity of the virus in sexual transmission (Zhang et al., 1993, 1996).
Because of the requirement for reverse transcription in the viral replication cycle, compounds that interfere with RT activity have been utilized as anti-HIV therapeutic agents. Many of these compounds, including 3′-azido-2′, 3′-dideoxythymidine (AZT), are nucleoside analogs that, upon activation by host cell kinases, are competitive inhibitors of reverse transcriptase (Furman et al., 1986). Other anti-RT compounds are nonnucleoside inhibitors (NNI), hydrophobic compounds that do not require cellular modification for antiviral activity. Examples of such compounds include nevirapine (Grob et al., 1992; Merluzzi et al., 1990), the pyridinones (Carroll et al., 1993; Goldman et al, 1991), and the carboxanilides (Bader et al, 1991; Balzarini et al, 1995, 1996). The nevirapine analog 9-azido-5,6-dihydro-11-ethyl-6-methyl-11H-pyrido[2,3b][1,5]benzodiazepin-5-one (9-AN) and the carboxanilide analog N-[4-chloro-3-(3 methyl-2-butenyloxy)phenyl]-2-methyl-3-furanocarbothiamide (UC781™) have been shown to be potent inhibitors of RT. In respect of the 9-AN, the exposure of a mixture of this compound and RT to UV-irradiation has been particularly effective in inhibiting RT. From the series of carboxanilides compounds, UC781™ has been found to be particularly effective(Barnard et al, 1997). The addition of a photoreactive label to UC781™ should increase further its ability to inactivate HIV RT, when a mixture of UC781™ and RT is exposed to UV-irradiation. The irradiation of a mixture of a photolabeled NNI of RT and RT is a type of photoinactivation.
The binding affinity and inhibitory effect of UC781™ is so high that the compound was able to eliminate HIV infectivity following short exposure of the isolated virus to UC781™ without the need for photoinactivation (Borkow et al, 1997). Further, this compound was shown to inhibit ERT in HIV virions and, when provided to HIV infected cells, caused the production of noninfectious nascent virus (Borkow et al., 1997). Therefore, it appears that, UC781™ is a particularly powerful inactivator of HIV. Although UC781™ has been proposed for use in retrovirucidal formulations (Borkow et al, 1997), use as a photoinactivator of HIV for the purpose of producing an non-infectious virus particle useful as an immunogen is absent from the prior art.
Non-infectious, virus-like particles have previously been produced via manipulations of the viral genome. For example, U.S. Pat. No. 6,080,408, incorporated herein by reference, discloses non-infectious virus-like particles wherein RT has been made inactive by virtue of deletions and sequence changes in the viral genome. Rovinski also discloses other methods of making non-infectious, virus-like particles, (or pseudovirions), that involve alterations to other genes of the HIV genome, expression of a subset of HIV virion components, and heat-inactivation of sera from HIV infected individuals. U.S. Pat. No. 6,017,543 discloses methods using formalin, psoralen, betapropriolactone, alone and in combination, as well as exposure to gamma radiation.
1.2.2 Immune Response to HIV
The immune response to HIV is composed of an initial cell mediated immune response followed by the subsequent development of neutralizing antibodies. Within weeks of infection, virus titers in the blood fall coincident with the induction of anti-HIV cellular and humoral immune responses. The fall in viremia correlates well with the appearance of anti-HIV major histocompatibility complex (MHC) class I-restricted CD8
+
cytotoxic T cells (Haynes et al., 1996). Recent evidence has shown a strong correlation of anti-HIV CD4
+
T cell responses and reduced viral loads (Rosenberg et al., 1997). Therefore, the presentation of HIV antigens in the context of MHC class II molecules to CD4+ T cells may be the key aspect of the control of the HIV infection.
Rosenberg et al. (1997) suggest that in HIV-1 infection, HIV-specific CD4
+
cells may be selectively eliminated. This may be due to the activation of these cells during high-level viremia, increasing their susceptibility to infection (Weissman et al., 1996; Stanley et al., 1996), or may be due to activation induced cell death during primary infection (Abbas, 1996). Nonetheless, increasing the virus-specific CD4
+
T cell response without infecting, or destroying, the responding cells may be an effective means of controlling viral loads. Therefore, some existing HIV vaccines may be ineffective because they do not provide presentation of HIV peptides in the context of MHC class II by antigen presenting cells.
1.2.3 HIV Vaccines
Historically, viral vaccines have been enormously successful in the prevention of infection by a particular virus. Therefore, when HIV was first isolated, there was a great amount of optimism that an HIV vaccine would be developed quickly. However, this optimism quickly faded because a number of unforeseen problems emerged. A discussion of the problems that an HIV vaccine must overcome is provided within Stott and Schild (1996) and is incorporated herein by reference.
First, HIV is a retrovirus, thus, during its growth cycle, proviral DNA is integrated in the hos
Fulbright & Jaworski L.L.P.
Park Hankyel T.
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